The integrity of the intestinal epithelium is essential to gut function and defense of the body against disease. Restitution, or migration to fill epithelial defects, requires motility of individual cells and it is thus important to understand the intracellular signaling mechanisms which regulate this process. This proposal focuses on phosphorylation and activation of the cytoplasmic tyrosine kinase Pyk2, which associates with membrane focal adhesions and is implicated in growth and cytoskeletal rearrangement. In intestinal epithelial cell (IEC) cultures, Pyk2 phosphorylation is strongly inducible and partially dependent on Rho and its effector Rho kinase (ROK). Inhibition of ROK alters the morphology of lECs and increases the area of migration into an artificial wound, leading to the hypothesis that Pyk2 plays a negative role. Therefore, the long-term objective of this application is to understand the roles of Pyk2 signaling and the Rho/ROK pathway in IEC migration, as well as to develop experimental models for correlating signaling events in cell culture (in vitro) with those in living animals (in vivo). The first Specific Aim is to characterize the relationship between Pyk2 and the Rho/ROK pathway during in vitro migration. We will examine the phosphorylation and localization of Pyk2 via immunofluorescent staining, and measure the rate of cell migration, while inhibiting ROK or altering Pyk2 expression. The second Specific Aim is to test the effects of ROK inhibition on Pyk2 phosphorylation, injury, and repair in a mouse intestinal wounding model. We will chemically induce colitis (via DSS), administer ROK inhibitor to the mice, then measure intestinal epithelial damage and assess Pyk2 activation using phospho-specific antibodies. By elucidating the signaling pathways involved in epithelial restitution, we will better understand the mechanisms underlying wound healing in the Gl epithelium; the development of in vivo models will support and validate in vitro data, and allow testing of potential therapeutic interventions. The potential impact of this project on public health lies in the promise of developing therapies for the modification of intestinal injury, either by preventing the development of epithelial wounds or promoting their closure. Furthermore, this research addresses basic mechanisms that may be conserved across different types of epithelium, such as mucous membranes, lung, and skin; it may thus prove relevant to wound healing in a range of traumatic, toxic, or infectious disease processes. [unreadable] [unreadable] [unreadable]